Many viruses use non-coding RNA elements to manipulate cellular machinery for effective infection and replication. Less frequently, elements are found in the coding region of a viral RNA. An example is the competitive inhibitor RNA (ciRNA) within the coding region of the C3 protease of group C enteroviruses, including coxsackievirus and poliovirus. This RNA 303 nucleotides element competitively inhibits RNase L, an RNase that becomes activated by the presence of double-stranded RNA in the cytoplasm. Once activated, RNase L rapidly degrades RNA and thus is a powerful antiviral enzyme. The ciRNA can serve to inhibit this enzyme, and this suggests there is strong selective pressure to maintain the ciRNA sequence to depress the antiviral response and facilitate successful infection. Understanding the detailed molecular events that occur during infection is important for the development of new therapies against poliovirus and coxsackievirus infection. These molecular event could also be important for a novel therapy were coxsackievirus A21 is in clinical trials as a therapy against melanoma, breast, and prostate cancers. The molecular, biophysical, and in vivo features of the ciRNA that inhibits RNase L are unknown; understanding the interactions between RNase L and ciRNA is important in understanding how an RNA is able to directly inhibit an enzyme that is made to ensure its destruction. Aim one of this proposal is t test the hypothesis that ciRNA folds into a unique structure when it binds making it able to inhibi RNAse L's ribonuclease activity, determine the binding interface between RNase L and ciRNA, and determine the molecular mechanism of inhibition within the ciRNA and RNase L complex by elucidating the structural interactions. To determine the features of this complex, I will characterize the importance of different secondary structural regions by chemical footprint probing, binding assays, and functional assays with wild-type and mutant forms of ciRNA with RNase L. Due the size of this complex I will focus on to characterize the structural relationship of the RNase L-ciRNA complex by X-ray crystallography. A structure of a complex between ciRNA and RNase L would illuminate whether inhibition is the result of encapsulating the active site, inducing a conformational change in the active site geometry of R Nase L directly, or another mechanism. The second aim of this proposal is to track the localization of RNase L and ciRNA during infection. I plan to use cell culture of monocytes, epithelial, and cancer cells and infect these cells with poliovirus and coxsackievirus strains to demonstrate the biological significance of the RNase L-ciRNA complex. RNase L and ciRNA will be fluorescently labeled track their movements during to invention to better understand their role within infection. The cell lines listed are being genetically edited by the CRIPR-Cas9 system to make GPF tagged RNase L and RNA FISH experiments will be used to track the ciRNA element. The combination of these experiments will allow to determine when and where RNase L and ciRNA co-localize.